In light of the importance of protein stability and orderly morphogenesis to lens function, the objective of this project is to determine the role of the multicatalytic proteinase complex (MPC; proteasome) in normal lens development and cataractogenesis. The approach is to establish the presence or absence in the lens of MPC and regulatory protein subunits with specific catalytic and regulatory functions; to determine the distribution of MPC and regulatory protein complexes and, where protein quantities are too small, their mRNAs among quiescent, proliferating and differentiating lens cells; to determine the effects of inhibitors of proteolysis on lens development; and to compare quantitatively and qualitatively the MPC and regulatory proteins and/or their mRNAs in normal and cataractous lenses. The experiments are designed to determine whether the 20S MPC or the 26S form, in which it is combined with the regulatory ATPase complex, plays a key role in differentiation or proliferation and if this role is modified in cataract. (i) Regulatory proteins will be purified by co-immunoprecipitation from rat and human lenses using affinity-purified monospecific anti-MPC antibody, and by column chromatography from the larger bovine lens. The immunoprecipitation experiments will allow us to compare the populations of MPC and regulatory proteins in different lenses and in different lens cell types by two- dimensional polyacrylamide gel electrophoresis. Purification of regulatory proteins will allow comparisons of their effects on MPC function and comparisons to homologous MPC subunits characterized in other organisms. (2) Individual subunit mRNAs will be quantitated to identify and characterize the MPC and regulatory subunits in normal rat lens, in rat lens explants induced to differentiate or proliferate and in cell populations from embryonic chick lens enriched for differentiating, proliferating or quiescent cells. (3) Inhibitor studies will determine the effect of proteinases on proliferation and differentiation in rat lens explants. (4) Collaborative immunocytochemistry experiments will be done to localize the MPC to specific cell types in rat and human lens. The MPC will be localized intracellularly to the cytosol or cell nucleus in rat lenses and in rat lens explants induced to proliferate or differentiate. The association of cytosolic MPC with a newly discovered cell fraction, the non-sedimentable lipid-protein microvesicle will be determined. (5) Sensitive specific reagents and techniques will be applied to compare MPC and the associated regulatory protein complexes in cataractous and normal rat and human lenses. These studies should enable identification of the specific MPC and regulatory protein populations present in the lens and the roles of this multifunctional enzyme system in lens development.